Although different nations have variable preferences for specific antidotes against cyanide poisoning, the antidotes of choice in the U.S. for over 30 years have been the combination of sodium nitrite and sodium thiosulfate, both administered intravenously in timely sequence. In recent years, this has been supplemented with the volatile amyl nitrite given nasally for its vasodilatory effect. However, these agents are generally not only slow acting, but have potential for toxicity and serious complications if not used properly. A recent perspective article on cyanide, authored by experts at the USAMRICD, pointed out that in severe cyanide poisonings, rapid intervention is the key, and treatments require a three minute solution, akin to the nerve agent antidote kit (Baskin et al. 2004). The availability of non-toxic agents that could be taken prophylactically by military personnel on threatened exposure or by first responders to a cyanide emergency, also represents an ideal requirement. These goals have not yet been achieved to date, and the present treatment modalities are unsuitable in a military setting, or in the event of actual use of cyanide as a threat agent on a large scale against the civilian population. We have recently developed (unpublished) a series of prototype cyanide antidotes that release the substrate for the enzyme, 3-mercaptopyruvate sulfur-transferase (3-MPST) in vivo, thereby providing a viable alternative method for detoxifying cyanide by utilizing this ubiquitous cellular enzyme to convert cyanide to the non-toxic thiocyanate. The rationale here is to provide this enzyme with its natural substrate directly, thereby by-passing the necessity for generating it endogenously from the transamination of L-cysteine, a sulfhydryl amino acid known to be less abundant in tissues. We have also developed a unique mouse model-that minimizes the use of large numbers of animals-for assessing the toxicity of sub-lethal doses of cyanide, which is highly amenable for evaluating the antidotal efficacy of our compounds. Having already established proof of concept that our prototype compounds protect against cyanide toxicity in mice, we will simultaneously a) expand the prototype series, and design and synthesize analogs around these series to improve antidotal efficacy, bioavailability, and physical properties of these compounds, b) superimpose the endogenous antioxidant glutathione (as its bioavailable form; vide infra) in the above regimen to evaluate whether such antioxidant co-treatment would improve survivability and protect against the neurological deficits seen in long term survivors of acute cyanide intoxication, and c) accelerate preclinical studies (acute and long term toxicity, ADME, other animal models, etc.) for those compounds already demonstrated to be protective, with the goal of filing an IND application to the FDA well within this grant period. Although most of the compounds of our series are rapid acting, at least one prototype (perhaps more) is slower acting (by design), but fully protective when administered orally; [unreadable] [unreadable] [unreadable]